Analytical method validation range

Validation is the determination of the attributes, or figures of merit, of an analytical method for one or more analytes in one or more sample matrices by one or more analysts in one or more analytical laboratories and the acceptance of the attributes as reasonable and useful by the users of the data. There are many levels of analytical method validation ranging from the validation of a method for a single analyte in a single matrix by a single analyst in a single laboratory to a multi-analyte, multi-matrix, multi-analyst, and multi-laboratory validation. 

Test methods used in the laboratory are generally derived from pharmacopeias such as the US Pharmacopoeia, British Pharmacopoeia or European Pharmacopoeia. For test methods that are not from recognized pharmacopoeias, validation of the analytical methods is required. The validation includes testing for accuracy, specificity, ruggedness, robustness, precision, detection limit, quantitation limit and range. A discussion of analytical methods validation is presented in Section 9.6.5. 

The process of method validation (i.e., evaluation of the assay) affects the quality of the quantitative data directly [9 A Guide to Analytical Method Validation, Waters Corporation]. Through method validation, it is assured that the method developed is acceptable. Issues involved in the validation of a mass spectrometry method for quantitative analysis are similar to those in any other analytical technique. The validation involves undertaking a series of studies to demonstrate the limit of detection G OD) limit of quantitation (LOQ) linear range specificity within-day precision and accuracy and day-to-day precision and accuracy, specificity, and robustness of the method. All of these parameters must be determined with those commonly accepted good laboratory practices criteria that are applicable in the vafidation of analytical methods. 

A subset of the dynamic range is the linear dynamic range. The linear dynamic range is that portion of the range for which the appropriate response function is a linear one. Historically, obtaining a linear calibration model has been the desired outcome of an analytical method development activity and most guidance for analytical method validations include an assessment of linearity (along with the... 

Other features of an analytical method that should be borne in mind are its linear range, which should be as large as possible to allow samples containing a wide range of analyte concentrations to be analysed without further manipulation, and its precision and accuracy. Method development and validation require all of these parameters to be studied and assessed quantitatively. 

To demonstrate the validity of an analytical method, data regarding working range/ calibration, recovery, repeatability, specificity and LOQ have to be provided for each relevant sample matrix. Most often these data have to be collected from several studies, e.g., from several validation reports of the developer of the method, the independent laboratory validation or the confirmatory method trials. If the intended use of a pesticide is not restricted to one matrix type and if residues are transferred via feedstuffs to animals and finally to foodstuffs of animal origin, up to 30 sets of the quality parameters described above are necessary for each analyte of the residue definition. Table 2 can be used as a checklist to monitor the completeness of required data. 

For multi-analyte and/or multi-matrix methods, it is not possible to validate a method for all combinations of analyte, concentration and type of sample matrix that may be encountered in subsequent use of the method. On the other hand, the standards EN1528 andEN 12393 consist of a range of old multi-residue methods. The working principles of these methods are accepted not only in Europe, but all over the world. Most often these methods are based on extractions with acetone, acetonitrile, ethyl acetate or n-hexane. Subsequent cleanup steps are based on solvent partition steps and size exclusion or adsorption chromatography on Florisil, silica gel or alumina. Each solvent and each cleanup step has been successfully applied to hundreds of pesticides and tested in countless method validation studies. The selectivity and sensitivity of GC combined with electron capture, nitrogen-phosphorus, flame photometric or mass spectrometric detectors for a large number of pesticides are acceptable. 

Verification implies that the laboratory investigates trueness and precision in particular. Elements which should be included in a full validation of an analytical method are specificity, calibration curve, precision between laboratories and/or precision within laboratories, trueness, measuring range, LOD, LOQ, robustness and sensitivity. The numbers of analyses required by the NMKL standard and the criteria for the adoption of quantitative methods are summarized in Table 10. 

If analytical methods are validated in inter-laboratory validation studies, documentation should follow the requirements of the harmonized protocol of lUPAC. " However, multi-matrix/multi-residue methods are applicable to hundreds of pesticides in dozens of commodities and have to be validated at several concentration levels. Any complete documentation of validation results is impossible in that case. Some performance characteristics, e.g., the specificity of analyte detection, an appropriate calibration range and sufficient detection sensitivity, are prerequisites for the determination of acceptable trueness and precision and their publication is less important. The LOD and LOQ depend on special instmmentation, analysts involved, time, batches of chemicals, etc., and cannot easily be reproduced. Therefore, these characteristics are less important. A practical, frequently applied alternative is the publication only of trueness (most often in terms of recovery) and precision for each analyte at each level. No consensus seems to exist as to whether these analyte-parameter sets should be documented, e.g., separately for each commodity or accumulated for all experiments done with the same analyte. In the latter case, the applicability of methods with regard to commodities can be documented in separate tables without performance characteristics. 

This analytical method provides very good precision and accuracy for the three parent herbicides over a 0.05-5.00 pgL range. Validation has been extended up to 20pgL-i. 

Method validation is needed to demonstrate the acceptability of the analytical method. A recovery test on a chemical being determined should be performed in order to verify the reliability of the series of analyses. Recovery studies are usually conducted by spiking untreated sediment with the target chemical at the deteetion limit, quantitation limit and in the range of 10-50 times the detection limit. The method is considered acceptable when the recoveries typically are greater than 70%. When the recovery is less than 70%, an improvement in the analytical methods is needed. Where this is not possible for technical reasons, then lower recovery levels may be acceptable provided that method validation has demonstrated that reproducible recoveries are obtained at a lower level of recovery. Analysis is usually done in duplicate or more, and the coefficient of variation (CV) should be less than 10% to ensure that recoveries will be consistently within the range 70-110%. 

For method tryout, run a control sample and two fortifications from each site. One fortification should be done at the LOQ and the other at the highest expected residue level, perhaps 1000 x LOQ. If the recoveries are within the acceptable range of 70-120% and there are no interferences, proceed with the method validation. If interferences are present which prevent quantitation of the analyte, try additional cleanup steps with SPE or use a more selective detection method such as liquid chromatography/mass spectrometry (LC/MS). 

For non-compendial procedures, the performance parameters that should be determined in validation studies include specificity/selectivity, linearity, accuracy, precision (repeatability and intermediate precision), detection limit (DL), quantitation limit (QL), range, ruggedness, and robustness [6]. Other method validation information, such as the stability of analytical sample preparations, degradation/ stress studies, legible reproductions of representative instrumental output, identification and characterization of possible impurities, should be included [7], The parameters that are required to be validated depend on the type of analyses, so therefore different test methods require different validation schemes. 

Few well characterized, validated methods are available for the determination of w-hexane in blood. A purge-and-trap method for volatiles has been developed and validated by researchers at the Centers for Disease Control and Prevention (CDC) (Ashley et al. 1992, 1994). Extension of the method to include /7-hexane should be possible. Current analytical methods utilize capillary GC columns and MS detection to provide the sensitivity and selectivity required for the analysis. Detection limits are in the low ppb range (Brugnone et al. 1991 Schuberth 1994). Headspace extraction followed by GC analysis has also been utilized for the determination of /7-hexanc in blood (Brugnone et al. 1991 Michael et al. 1980 Schuberth 1994) however, very little performance data are available. 

Method validation is the process of proving that an analytical method is acceptable for its intended purpose. Many organizations provide a framework for performing such validations (ASTM, 2004). In general, methods for product specifications and regulatory submission must include studies on specificity, linearity, accuracy, precision, range, detection limit, and quantitation limit. 

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